The presently disclosed and claimed inventive concepts are related to, but not limited to, substrates and inhibitors of α2-antiplasmin cleaving enzyme and fibroblast activation protein-alpha and to screening methods for identifying such inhibitors, and to methods for treating conditions involving fibrin and α2-antiplasmin, including plaque and clot formation, atherosclerosis and cancers involving fibroblast activation protein.
α2-Antiplasmin (α2AP) is a glycoprotein in blood plasma that rapidly and specifically inhibits the enzyme, plasmin, which digests blood clots, whether presenting early as intravascular platelet-fibrin deposits or as partially or completely occlusive thrombi. Similarly, plasmin and α2AP activities are important to the development and survival of fibrin as occurs in inflammation, wound healing and virtually all forms of cancer and its metastases.
Human α2-antiplasmin (α2AP), also known as α2-plasmin inhibitor, is the main inhibitor of plasmin. Plasmin plays a critical role in fibrin proteolysis and tissue remodeling. The physiologic relevance of plasmin inhibition by α2AP to blood clotting and fibrinolytic homeostasis is supported by the following observations: (1) the rate of free plasmin inactivation by circulating α2AP is much faster than fibrin(ogen) digestion by plasmin, thereby eliminating the possibility of a systemic lytic state and consequent bleeding; (2) α2AP is cross-linked to forming fibrin by activated blood clotting factor XIII (FXIIIa) and inhibits plasmin-mediated lysis in direct proportion to the amount incorporated; and (3) patients with homozygous α2AP deficiency manifest serious hemorrhagic tendencies, while heterozygotes tend to bleed only after major trauma or surgery. Human α2AP is synthesized primarily in the liver, and during circulation in plasma, the secreted precursive form, Met-α2-antiplasmin (Met-α2AP), a 464-residue protein having methionine as the N-terminus, undergoes proteolytic cleavage between Pro12 and Asn13 (the P1-P1′ scissile bond) to yield Asn-α2-antiplasmin (Asn-α2AP), a 452-residue version with asparagine as the N-terminus. Met-α2AP accounts for approximately 30% of circulating α2AP, and Asn-α2AP accounts for approximately 70%.
When the Met-form of α2AP was found in plasma and its gene sequenced, there initially appeared to be a discrepancy in one of the nucleotides encoding the sixth amino acid. Two groups found a cytidine (C) resulting in Arg as the sixth amino acid, and one group found thymidine (T), resulting in Trp at that position. It was suggested that the difference was due to one group having used liver carcinoma cells as a source of DNA, while the other two groups used normal cells. It has now been determined that both Arg6 and Trp6 forms of Met-α2AP exist in normal human plasma samples. An investigation of a mutant α2AP in a family with bleeding tendencies identified the mutation responsible for the ineffective α2AP along with three polymorphisms in the α2AP gene including this C/T single nucleotide polymorphism (SNP); this study examined 30 normal blood donors and reported an allelic frequency of 0.81/0.19 for the C/T SNP. No larger studies of a normal population have been done to examine the frequency of homozygotes and heterozygotes, or whether genotype might affect ratios of Met- to Asn-α2AP in plasma. The Arg6Trp SNP was apparently assumed to be a silent polymorphism, but biochemical examination of the two polymorphic forms of Met-α2AP on yielding the derivative form, Asn-α2AP, its incorporation into fibrin and the impact on fibrinolysis have never been assessed prior to the present work.
We discovered antiplasmin-cleaving enzyme (APCE) in human plasma and showed that it is a soluble isoform or derivative of fibroblast activation protein-alpha (FAP), the latter being a type II integral membrane protein, which is predicted to have its first six N-terminal residues within fibroblast cytoplasm, followed by a 20-residue transmembrane domain, and then a 734-residue extracellular C-terminal catalytic domain. Like APCE, FAP is also a prolyl-specific enzyme that exhibits both endopeptidase and dipeptidyl peptidase activities. FAP is expressed by activated fibroblasts during embryogenesis, wound healing and expansion of epithelial-derived cancers, but not by normal tissues. We have also reported that FAP and APCE are essentially identical in amino acid sequence, except that APCE lacks the first 23 amino terminal residues of FAP, but otherwise the two molecules have essentially identical physico-chemical properties.
Specific inhibitors of APCE which result in enhanced endogenous fibrinolytic dispersion of intravascular platelet-fibrin thrombi, or that block FAP and its proteolysis of extracellular matrix during cancers expansion or other conditions of abnormal cell proliferation are desirable and are objects of the presently disclosed and claimed inventive concepts.